We have recently discovered a novel "Subtilase cytotoxin" produced by a strain of Shiga toxigenic Escherichia coli (a category B Priority Pathogen) that was responsible for an outbreak of hemolytic uremic syndrome (HUS). It belongs to a new class of AB5 cytotoxins, because its A subunit has distinct enzymic activity and it has no sequence similarity with any of the other AB5 toxin families (cholera, Shiga and pertussis toxins). Subtilase cytotoxin is extraordinarily cytotoxic for cultured cells and is lethal for mice (with histopathology similar to that of HUS). Apart from its possible role in severe human disease, it is a potential bioterrorism agent which could have global impact. The long-term objective of this response to PA-04-119 is complete structural and functional characterization of the emerging toxin. Its Specific Aims are: 1. Elucidation of the molecular basis for cytotoxicity. A subunit Subtilase-like serine protease activity is essential for cytotoxicity, and preliminary data indicate that the ER chaperone BiP is a key cellular target for the toxin. In this Aim, we will continue our investigation of the molecular basis for cytotoxicity by examining the downstream consequences of BiP cleavage, including whether this results in ER stress and apoptosis. 2. Investigation of toxin trafficking in target cells. We hypothesize that after binding to the cell surface, the toxin must undergo retrograde transport to the ER in order to exert its toxic effects. Intracellular toxin trafficking will therefore be examined by co-localization of labelled toxin with cellular compartmental markers using confocal microscopy. 3. Examination of tissue tropism and in vivo effects of the toxin. The distribution of toxin receptors in mouse tissues and trafficking of labelled toxin in vivo will be examined using fluorescence, confocal and electron microscopy. The direct contribution of Subtilase cytotoxin to the pathogenesis of STEC disease will also be investigated in a mouse model. 4. Determination of the 3Dstructure of the toxin. X-ray crystallography and computer modelling will be used to solve the 3D structure. Relevance: This project will provide essential information on the basic biology of this emerging toxin, particularly its structure and the mechanism whereby it damages cells and tissues. This will provide a foundation for the future development of countermeasures including therapeutic agents and vaccines.